1. Field of the Invention
The present invention relates generally to the fields of molecular biology, cancer biology, and gene therapy. More particularly, the invention concerns compositions comprising and methods utilizing novel promoters that include a tissue-selective promoter sequence and a second promoter sequence operatively coupled to the tissue-selective promoter sequence, wherein the second promoter sequence includes a minimal promoter sequence, preferably a minimal viral promoter sequence.
2. Description of Related Art
One of the major obstacles to successful gene therapy is the lack of an effective delivery system that can be targeted to a tissue of interest. For example, it would be beneficial to be able to target gene therapy for cancer to the tumor in the cancer patient. One means for tumor-targeted transgene expression is to control gene expression through the use of promoters that are selective for tumors.
One such promoter is the human telomerase reverse transcriptase (hTERT) promoter. hTERT is the catalytic subunit of telomerase. Telomerase is a ribonucleoprotein complex that is responsible for the complete replication of chromosomal ends (Blackburn, 1991). Many studies have demonstrated that the majority of malignant tumors express telomerase activity (Kim et al., 1996), whereas most normal cells do not (Shay and Wright, 1996). Three major components associated with telomerase activity in humans have been identified: (a) the RNA component [hTER (Feng et al., 1995)]; (b) the telomerase-associated protein [hTEP1 (Harrington et al., 1997)]; and (c) the telomerase catalytic unit or human telomerase reverse transcriptase [hTERT (Meyerson et al., 1997; Nakamura et al., 1997)]. Only hTER and hTERT, however, are required for the reconstitution of telomerase activity in vitro (Nakayama et al., 1998) and therefore represent the minimal catalytic core of telomerase in humans (Beattie et al., 1998).
The promoter region of hTERT has been cloned and characterized previously (Takakura et al., 1999). Telomerase-specific expression of cytotoxic or proapoptotic genes such as the diphtheria toxin A-chain, FADD, caspases, and Bax by the hTERT promoter has been successfully achieved and reporter in various gene transfer systems, such as plasmid and adenovirus (see, e.g., Abdul-Ghani et al., 2000; and Komata et al., 2001).
However, the transcription promoting strength of the hTERT promoter, like most other intrinsic mammalian promoters, is usually much weaker than commonly used viral promoters such as the CMV promoter and the SV40 early promoter. Consequently, it use for gene therapy is hampered by the problem of low transgene expression.
To overcome the weak transcription promoting capability of unmodififed hTERT promoter in mammalian cells in vitro and in vivo, some investigators have developed a binary adenoviral vector system wherein the hTERT promoter and transgene are placed under the control of the Ga14 gene or tetracycline responsive element (TRE) in a first vector (Gu et al., 2000; Gu et al., 2002). A second vector expresses an enhancer such as VP16 protein or Tet-On/Tet-Off transactivators to augment transgene expression from hTERT. However, the system is too complex and impractical for clinical application because of the deficiencies associated with the use of two separate vectors. In particular, the dual vector system is random and uncontrollable because it is difficult for both vectors to enter the same cell at the same time. Furthermore, it may potentially result in increased toxicity due to the use of multiple vectors and multiple therapeutically unrelated components and gene products involved in the system.
Therefore, there is the need for more effective tissue-selective promoters or improved methods of facilitating promoter function to allow for enhanced tissue-selective transgene expression. Promoter technology in this area would facilitate the clinical application and development of vectors for use in gene therapy as well as other technologies that require high transgene expression, such as reporter-based imaging modalities. Combining this technology into a single vector would help to diminish the toxicity associated with dual or multiple vectors.